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WO2002043767A1 - Apport oral ameliore de peptides au moyen de translocateurs de membrane pouvant etre coupes par une enzyme - Google Patents

Apport oral ameliore de peptides au moyen de translocateurs de membrane pouvant etre coupes par une enzyme Download PDF

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Publication number
WO2002043767A1
WO2002043767A1 PCT/US2001/044980 US0144980W WO0243767A1 WO 2002043767 A1 WO2002043767 A1 WO 2002043767A1 US 0144980 W US0144980 W US 0144980W WO 0243767 A1 WO0243767 A1 WO 0243767A1
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Prior art keywords
pharmaceutical composition
peptide
agent
lowering agent
acid
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PCT/US2001/044980
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English (en)
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WO2002043767A8 (fr
Inventor
William Stern
Nozar M. Mehta
Martha V.L. Ray
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Unigene Laboratories Inc.
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Priority to JP2002545737A priority Critical patent/JP4476544B6/ja
Priority to CA2430366A priority patent/CA2430366C/fr
Priority to ES01996021.0T priority patent/ES2642628T3/es
Priority to DK01996021.0T priority patent/DK1339431T3/en
Priority to AU2706002A priority patent/AU2706002A/xx
Priority to EP01996021.0A priority patent/EP1339431B1/fr
Publication of WO2002043767A1 publication Critical patent/WO2002043767A1/fr
Publication of WO2002043767A8 publication Critical patent/WO2002043767A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4891—Coated capsules; Multilayered drug free capsule shells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/585—Calcitonins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention relates to oral peptide pharmaceuticals where the active compounds include a plurality of amino acids and at least one peptide bond in their molecular structures, and -to methods of enhancing bioavailability of such peptide active compounds when administered orally.
  • peptides Numerous human hormones, neurotransmitters and other important biological compounds have peptides as a substantial part of their molecular structures. Many diseases respond positively to raising the level of these peptide compounds in patients. Therapeutically effective amounts of such biologically relevant peptides may be administered to patients in a variety of ways. However, as discussed further below, preferred oral administration is very difficult with this type of active compound.
  • Salmon calcitonin for example, is a peptide hormone which decreases calcium release from bone. When used to treat bone-related diseases and calcium disorders (such as osteoporosis, Paget ' s disease, hypercalcemia of malignancy, and the like) , it has the effect of helping maintain bone density. Many types of calcitonin have been isolated (human calcitonin, salmon calcitonin, eel calcitonin, elkatonin, porcine calcitonin, and chicken calcitonin) . There is significant structural non-homology among the various calcitonin types. For example, there is only 50% percent identity between the amino acids making up human calcitonin and those making up salmon calcitonin. Notwithstanding the difference in molecular structure, salmon calcitonin may be used in the human treatment of the calcitonin-responsive diseases discussed above.
  • Peptide pharmaceuticals used in the prior art frequently have been administered by injection or by nasal administration.
  • Insulin is one example of a peptide pharmaceutical frequently administered by injection.
  • a more preferred oral administration tends to be problematic because peptide active compounds are very susceptible to degradation in the stomach and intestines.
  • Salmon calcitonin lacks sufficient stability in the gastrointestinal tract, and tends to be poorly transported through intestinal walls into the blood.
  • injection and nasal administration are significantly less convenient than, and involve more patient discomfort than, oral administration. Often this inconvenience or discomfort results in substantial patient noncompliance with a treatment regimen.
  • Proteolytic enzymes of both the stomach and intestines may degrade peptides, rendering them inactive before they can be absorbed into the bloodstream. Any amount of peptide that survives proteolytic degradation by proteases of the stomach
  • the plasma membrane of eukaryotic cells is impermeable to large peptides or proteins.
  • certain hydrophobic amino acid sequences variously called as ferry peptides or membrane translocating sequences, when fused to the N- or C- terminus of functional proteins, can act as membrane translocators, and mediate the transport of these proteins into living cells.
  • This method of protein delivery into cells while potentially very useful, has two main drawbacks. First, the protein cannot be targeted to any specific cell type. Therefore, once it is injected and enters the circulation, it will presumably enter all cell types in a non-specific, non-receptor mediated manner.
  • pharmaceutical peptides e.g., physiologically active peptide agents such as insulin, salmon calcitonin, vasopressin and others discussed herein.
  • the invention provides a pharmaceutical composition for oral delivery of a physiologically active peptide agent comprising:
  • Preferred peptide active agents include but are not limited to insulin, vasopressin salmon calcitonin, glucagon-like peptide 1, parathyroid hormone, luteinizing hormone releasing hormone, erythropoeitin, and analogs thereof. Especially preferred is salmon calcitonin.
  • the invention provides a method for enhancing the bioavailability of a therapeutic peptide active agent delivered orally, said method comprising:
  • the present invention is believed to reduce the likelihood of proteolytic degradation of the peptide active compound by simultaneously protecting the peptide from proteolytic attack by (1) stomach proteases which are typically most active at acidic pHs and (2) intestinal or pancreatic proteases (which are typically most active at basic to neutral pH) .
  • the invention is believed to promote the process by which the peptide crosses the intestinal brush border membrane into the blood due to the presence of the membrane translocator, while continuing to protect the peptide from proteolytic degradation.
  • An acid resistant protective vehicle protects the peptide active agent from the acid-acting proteases of the stomach. Significant quantities of acid (with which the peptide active agent is intermixed) then reduce the activity of neutral to basic-acting proteases in the intestine (e.g., luminal or digestive protease and proteases of the brush border membrane) by lowering pH below the optimal activity range of these intestinal proteases.
  • the membrane translocator capable when linked to the active peptide agent enhances transport of the peptide agent through intestinal mucous layers, through the brush border membrane and into the blood.
  • the membrane translocator is cleaved by a blood or lymphatic system protease, thus releasing the active peptide agent in a patient's system.
  • Figure 1 shows the circular plasmid map of the Universal Cloning Vector pUSEC-05.
  • Figure 2 shows the circular plasmid map of pMT3- sCT-01, a vector for the expression of the MT-sCT fusion sequence.
  • Figure 3 shows the circular plasmid map of the pSCT025 vector containing the sCT gene.
  • Figure 4 shows the circular plasmid map of the Universal Cloning Vector pUSEC-06.
  • Figure 5 shows the circular plasmid map of pMT3- sCT-02, a vector for the expression of the MT-sCT fusion sequence.
  • Figure 6 shows the circular plasmid map of pMT3- sCT-03, a vector for the expression of the MT-sCT fusion sequence.
  • Figure 7 shows a 10-20 % gradient SDS-PAGE gel for the following samples:
  • Lane 3 Insoluble cell lysate material (purified recombinant OmpA-MT3-sCTgly) , input to purification; Lane 4 Analytical RP analysis of solubilization;
  • Lane 5 Preparative purification sample post solubilization, 4uL
  • Lane 6 Preparative purification sample post solubilization, 1.5 uL
  • Lane 4 Analytical RP analysis of solubilization
  • Lane 5 Preparative purification sample post solubilization, 4uL
  • patients in need of treatment with peptide active ingredients are provided with an oral pharmaceutical composition thereof (at appropriate dosage) , preferably but not necessarily in tablet or capsule form of an ordinary size in the pharmaceutical industry.
  • oral pharmaceutical composition thereof at appropriate dosage
  • Patients who may benefit are any who suffer from disorders that respond favorably to increased levels of a peptide-containing compound.
  • oral salmon calcitonin in accordance with the invention may be used to treat patients who suffer from calcium disorders or bone diseases.
  • the invention may be used, for example, to treat osteoporosis, Paget ' s disease, hypercalcemia of malignancy and the like, with oral calcitonin, preferably salmon calcitonin.
  • Salmon calcitonin is a preferred active ingredient for use in accordance with the invention for a number of reasons. For example, it provides a number of advantages over even human calcitonin, even though used as a pharmaceutical agent for human patients. Among the advantages provided by utilizing salmon calcitonin instead of human calcitonin for the treatment of human osteoporosis are increased potency, analgesia and increased half-life. Salmon calcitonin is more effective than natural human calcitonin in treatment, since lower dosages are necessary than with human calcitonin. There is substantial non-homology between salmon and human calcitonin, with only 50% identity in the amino acid sequences of the two calcitonins.
  • Salmon calcitonin enjoys an unexpectedly higher bioavailability when administered orally in accordance with the present invention than would be expected for its molecular weight.
  • bioavailability of salmon calcitonin when linked to a membrane translocator (MT) according to the invention is significantly increased.
  • the pharmaceutical composition of the invention is believed to overcome a series of different and unrelated natural barriers to bioavailability.
  • Various components of the pharmaceutical compositions act to overcome different barriers by mechanisms appropriate to each, and result in synergistic effects on the bioavailability of a peptide active ingredient.
  • the peptide active compound may be administered orally.
  • the presence of at least one MT, preferably two MTs, more preferably, two peptide MTs would enhance the membrane permeability of the fusion peptide across the lumen of the intestine and provide for improved bioavailability. Since the MT link to the active peptide can be cleaved by an enzyme in the blood or the lymphatic system, thereby leaving the active peptide free to reach its target.
  • proteolytic degradation of the peptide and of the membrane translocator by stomach enzymes most of which are active in the acid pH range
  • intestinal or pancreatic proteases most of which are active in the neutral to basic pH range
  • the peptide is transported through the stomach under the protection of an appropriate acid-resistant protective vehicle for substantially preventing contact between the salmon calcitonin or other active peptide and any stomach proteases capable of degrading it .
  • an appropriate acid-resistant protective vehicle for substantially preventing contact between the salmon calcitonin or other active peptide and any stomach proteases capable of degrading it .
  • the acid is believed to lower the local intestinal pH (where the active agent has been released) to levels below the optimal range for many intestinal proteases and other intestinal enzymes. This decrease in pH reduces the proteolytic activity of the intestinal proteases, thus affording protection to the peptide and the membrane translocator from potential degradation.
  • the activity of these proteases is diminished by the temporarily acidic environment provided by the invention. It is preferred that sufficient acid be provided that local intestinal pH is lowered temporarily to 5.5 or below, preferably 4.7 or below and more preferably 3.5 or below.
  • the sodium bicarbonate test described below is indicative of the required acid amount.
  • conditions of reduced intestinal pH persist for a time period sufficient to protect the peptide agent and the membrane translocator from proteolytic degradation until at least some of the peptide agent has had an opportunity to cross the intestinal wall into the bloodstream.
  • For salmon calcitonin experiments have demonstrated a T raax of 5-15 minutes for blood levels of salmon calcitonin when the active components are injected directly into the duodenum, ileum or colon of rats.
  • protease inhibitors are believed to reduce the proteolytic activity of the intestinal proteases, thus affording protection to the peptide and the membrane translocator from premature potential degradation.
  • compositions of the present invention can optionally contain absorption enhancers.
  • the absorption enhancers of the invention synergistically promote peptide absorption into the blood while conditions of reduced proteolytic activity prevail .
  • the mechanism by which the invention is believed to accomplish the goal of enhanced bioavailability is aided by having active components of the pharmaceutical composition released together as simultaneously as possible.
  • enteric coating is less likely to interfere with peptide release, or with the release of other components in close time proximity with the peptide.
  • the enteric coating should normally add less than 30% to the weight of the remainder of pharmaceutical composition (i.e., the other components of the composition excluding enteric coating) .
  • it is less than 20% and, more preferably, the enteric coating adds between 10% and 20% to the weight of the uncoated ingredients.
  • the absorption enhancer which may be a solubility enhancer and/or transport enhancer (as described in more detail below) aids transport of the peptide agent from the intestine to the blood, and may promote the process so that it better occurs during the time period of reduced intestinal pH and reduced intestinal proteolytic activity.
  • Many surface active agents may act as both solubility enhancers and transport (uptake) enhancers.
  • enhancing solubility provides (1) a more simultaneous release of the active components of the invention into the aqueous portion of the intestine, (2) better solubility of the peptide in, and transport through, a mucous layer along the intestinal walls.
  • an uptake enhancer provides better transport through the brush border membrane of the intestine into the blood, via either transcellular or paracellular transport .
  • many preferred compounds may provide both functions. In those instances, preferred embodiments utilizing both of these functions may do so by adding only one additional compound to the pharmaceutical composition. In other embodiments, separate absorption enhancers may provide the two functions separately.
  • Peptide active ingredients which may benefit from oral delivery in accordance with the invention include any therapeutic agent that is physiologically active and has a plurality of amino acids and at least one peptide bond in its molecular structure. These peptide active ingredients are linked to an MT sequence to facilitate their absorption from the intestine. The MT must be protected from cleavage by proteases in the stomach and intestine before its absorption. However, once absorbed, the MT should be able to be at least partially removed by proteases to free up the active peptide.
  • the MT can comprise an amino acid sequence, preferably a signal peptide or signal sequence.
  • a "signal peptide, " as used herein, is a sequence of amino acids generally but not necessarily of a length of about 10 to about 50 or more amino acid residues, many (typically about 55-60%) • residues of which are hydrophobic such that they have a hydrophobic, lipid-soluble portion. The hydrophobic portion is a common, major motif of the signal peptide, and it is often a central part of the signal peptide of protein secreted from cells.
  • a signal peptide is a peptide capable of penetrating through the cell membrane to allow the export of cellular proteins.
  • the signal peptides of this invention are also "importation competent,” i.e., capable of penetrating through the cell membrane from outside the cell to the interior of the cell.
  • the amino acid residues can be mutated and/or modified (i.e., to form mimetics) so long as the modifications do not affect the translocation- mediating function of the peptide.
  • the word "peptide” includes mimetics and the word “amino acid” includes modified amino acids, as used herein, unusual amino acids, and D-form amino acids.
  • All importation competent signal peptides encompassed by this invention have the function of mediating translocation across a cell membrane from outside the cell to the interior of the cell. They may also retain their ability to allow the export of a protein from the cell into the external milieu. A putative signal peptide can easily be tested for this importation activity following the teachings provided herein, including testing for specificity for any selected cell type.
  • the MT can also comprise fatty acids and/or bile acids.
  • Such molecules when used, are linked to the active peptide by an amino acid bridge which is subject to cleavage by proteases in the plasma.
  • the MT can be linked to the active peptide by a non- peptidyl linkage, in which case the in vivo enzyme that cleaves the linkage may be an enzyme other than protease.
  • the amino acid bridge must be a target for cleavage by at least one plasma protease. Plasma proteases as well as their target sequences are well known in the art. Table 2 illustrates some of these enzymes as well as their specific targets Table 2 - Plasma Proteases and their Specific Targets
  • the invention by several mechanisms, suppresses the degradation of -the active ingredient linked to an MT by protease that would otherwise tend to cleave one or more of the peptide bonds of the active ingredient .
  • the molecular structure of the active ingredient may further include other substituents or modifications.
  • salmon calcitonin a preferred peptide active agent herein, can be amidated at its C-terminus. Both synthetic and natural peptides can be orally delivered in accordance with the invention.
  • Peptide active compounds of the invention include, but are not limited to, insulin, vasopressin, calcitonin (including not only the preferred salmon calcitonin, but other calcitonins as well) .
  • Other examples include calcitonin gene-related peptide, parathyroid hormone, luteinizing hormone-releasing factor, erythropoietin, tissue plasminogen activators, human growth hormone, adrenocorticototropin, various interleukins, enkephalin, glucagon-like peptide 1, and all analogs thereof. Many others are known in the art.
  • any pharmaceutical compound having peptide bonds which would be subject to cleavage in the gastrointestinal tract would benefit from oral delivery in accordance with the present invention because of the enhancement of absorption of such compounds from the intestine coupled with the reduction in such cleavage that is afforded by the present invention.
  • Salmon calcitonin When salmon calcitonin is used, it preferably comprises from 0.02 to 0.2 percent by weight relative to the total weight of the overall pharmaceutical composition (exclusive of enteric coating) Salmon calcitonin is commercially available (for example, from BACHEM, Torrence, California) . Alternatively it may be synthesized by known methods, some of which are discussed briefly below. Other peptide active agents should be present at higher or lower concentrations depending on desired target blood concentrations for the active compound and its bioavailability in the oral delivery system of the invention.
  • Salmon calcitonin precursors may be made by either chemical or recombinant syntheses known in the art. Precursors of other amidated peptide active agents may be made in like manner. Recombinant production is believed to be significantly more cost effective. Precursors are converted to active salmon calcitonin by amidation reactions that are also known in the art. For example, enzymatic amidation is described in U.S. Patent 4,708,934 and European Patent Publications 0 308 067 and 0 382 403. Recombinant production is preferred for both the precursor and the enzyme that catalyzes the conversion of the precursor to salmon calcitonin. Such recombinant production is discussed in Biotechnology, Vol. 11 (1993) pp. 64-70, which further describes a conversion of a precursor to an amidated product . The recombinant product reported there is identical to natural salmon calcitonin, and to salmon calcitonin produced using solution and solid phase chemical peptide synthesis.
  • linking as used herein is meant that the biologically active peptide is associated with the MT in such a manner that when the MT crosses the cell membrane, the active peptide is also imported across the cell membrane.
  • Examples of such means of linking include (A) linking the MT to the active peptide by a peptide bond, i.e., the two peptides (the peptide part of the MT and the active peptide) can be synthesized contiguously; (B) linking the MT to the active peptide by a non-peptide covalent bond (such as conjugating a signal peptide to a protein with a crosslinking reagent) ; (C) chemical ligation methods can be employed to create a covalent bond between the carboxy-terminal amino acid of an MT such as a signal peptide and the active peptide.
  • a peptide is synthesized, by standard means known in the art, (Merrifield, J. Am. Chem. Soc . 85:2149-2154, 1963; and Lin et al . , Biochemistry 27:5640-5645, 1988) and contains, in linear order from the amino-terminal end, a signal peptide sequence (the MT) , an amino acid sequence that can be cleaved by a plasma protease, and a biologically active amino acid sequence.
  • a signal peptide sequence the MT
  • Such a peptide could also be produced through recombinant DNA techniques, expressed from a recombinant construct encoding the above-described amino acids to create the peptide. (Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) .
  • a peptide bond as above, can be utilized or a non-peptide covalent bond can be used to link the MT with the biologically t active peptide, polypeptide or protein.
  • This non-peptide covalent bond can be formed by methods standard, in the art, such as by conjugating the MT to the peptide, polypeptide or protein via a crosslinking reagent, for example, glutaraldehyde . Such methods are standard in the art . (Walter et al . , Proc. Natl. Acad. Sci. USA 77:5197; 1980).
  • the production of the preferred recombinant salmon calcitonin may proceed, for example, by producing glycine-extended salmon calcitonin precursor in E. coli as a soluble fusion protein with glutathione-S- transferase.
  • the glycine-extended precursor has a molecular structure that is identical to active salmon calcitonin except at the C-terminal (where salmon calcitonin terminates -pro-NH 2 , while the precursor terminates -pro-gly.
  • An ⁇ -amidating enzyme described in the publications above catalyzes conversion of precursors to salmon calcitonin.
  • That enzyme is preferably recombinantly produced, for example, in Chinese Hamster Ovary (CHO) cells) as described in the Biotechnology article cited above.
  • Other precursors to other amidated peptides may be produced in like manner.
  • Peptides that do not require amidation or other additional functionalities may also be produced in like manner.
  • Other peptide active agents are commercially available or may be produced by techniques known in the art .
  • the total amount of the pH-lowering compound to be administered with each administration of salmon calcitonin should preferably be an amount which, when it is released into the intestine, is sufficient to lower the local intestinal pH substantially below the pH optima for proteases found there.
  • the quantity required will necessarily vary with several factors including the type of pH-lowering agent used (discussed below) and the equivalents of protons provided by a given pH-lowering agent.
  • the amount required to provide good bioavailability is an amount which, when added to a solution of 10 milliliters of 0.1 M sodium bicarbonate, lowers the pH of that sodium bicarbonate solution to no higher than 5.5, and preferably no higher than 4.7, most preferably no higher than 3.5.
  • Enough acid to lower pH, in the foregoing test, to about 2.8 may been used in some embodiments.
  • Preferably at least 300 milligrams, and more preferably at least 400 milligrams of the pH- lowering agent are used in the pharmaceutical composition of the invention.
  • the foregoing preferences relate to the total combined weight of all pH-lowering agents where two or more of such agents are used in combination.
  • the oral formulation should not include an amount of any base which, when released together with the pH-lowering compound, would prevent the pH of the above-described sodium bicarbonate test from dropping to 5.5 or below.
  • the pH-lowering agent of the invention may be any pharmaceutically acceptable compound that is not toxic in the gastrointestinal tract and is capable of either delivering hydrogen ions (a traditional acid) or of inducing higher hydrogen ion content from the local environment. It may also be any combination of such compounds. It is preferred that at least one pH-lowering agent used in the invention have a pKa no higher than 4.2, and preferably no higher than 3.0. It is also preferred that the pH lowering agent have a solubility in water of at least 30 grams per 100 milliliters of water at room temperature .
  • Examples of compounds that induce higher hydrogen ion content include aluminum chloride and zinc chloride.
  • Pharmaceutically acceptable traditional acids include, but are not limited to acid salts of amino acids (e.g. amino acid hydrochlorides) or derivatives thereof. Examples of these are acid salts of acetylglutamic acid, alanine, arginine, asparagine, aspartic acid, betaine, carnitine, carnosine, citrulline, creatine, glutamic acid, glycine, histidine, hydroxylysine, hydroxyproline, hypotaurine, isoleucine, leucine, lysine, methylhistidine, norleucine, ornithine, phenylalanine, proline, sarcosine, serine, taurine, threonine, tryptophan, tyrosine and valine.
  • pH-lowering compounds include carboxylic acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyceric, glycocolic, glyoxylic, isocitric, isovaleric, lactic, maleic, oxaloacetic, oxalosuccinic, propionic, pyruvic, succinic, tartaric, valeric, and the like.
  • carboxylic acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyceric, glycocolic, glyoxylic, isocitric, isovaleric, lactic, maleic, oxaloacetic, oxalosuccinic, propionic, pyruvic, succinic, tartaric, valeric, and the like.
  • pH-lowering agents that might not usually be called “acids” in the art, but which may nonetheless be useful in accordance with the invention are phosphate esters (e.g., fructose 1, 6 diphosphate, glucose 1, 6 diphosphate, phosphoglyceric acid, and diphosphoglyceric acid) .
  • phosphate esters e.g., fructose 1, 6 diphosphate, glucose 1, 6 diphosphate, phosphoglyceric acid, and diphosphoglyceric acid
  • CARBOPOL ® Trademark BF Goodrich
  • polymers such as polycarboph.il may also be used to lower pH.
  • pH lowering agent that achieves the required pH level of no higher than 5.5 in the sodium bicarbonate test discussed above may be used.
  • One preferred embodiment utilizes, as at least one of the pH- lowering agents of the pharmaceutical composition, an acid selected from the group consisting of citric acid, tartaric acid and an acid salt of an amino acid.
  • pH- lowering agents An alternative or a supplement to the use of pH- lowering agents is the use of protease inhibitors, in particular inhibitors of intestinal proteases.
  • protease inhibitors in particular inhibitors of intestinal proteases.
  • Table 3 illustrates some of the known intestinal proteases.
  • the absorption enhancers are preferably present in a quantity that constitutes from 0.1 to 20.0 percent by weight, relative to the overall weight of the pharmaceutical composition (exclusive of the enteric coating) .
  • Preferred absorption enhancers are surface active agents which act both as solubility enhancers and uptake enhancers.
  • solubility enhancers improve the ability of the components of the invention to be solubilized in either the aqueous environment into which they are originally released or into the lipophilic environment of the mucous layer lining the intestinal walls, or both.
  • (uptake) enhancers are those which facilitate the ease by which peptide agents cross the intestinal wall .
  • One or more absorption enhancers may perform one function only (e.g., solubility), or one or more absorption enhancers may perform the other function only (e.g., uptake), within the scope of the invention. It is also possible to have a mixture of several compounds some of which provide improved solubility, some of which provide improved uptake and/or some of which perform both. Without intending to be bound by theory, it is believed that uptake enhancers may act by (1) increasing disorder of the hydrophobic region of the membrane exterior of intestinal cells, allowing for increased transcellular transport; or (2) leaching membrane proteins resulting in increased transcellular transport; or (3) widening pore radius between cells for increased paracellular transport .
  • detergents are useful in (1) solubilizing all of the active components quickly into the aqueous environment where they are originally released, (2) enhancing lipophilicity of the components of the invention, especially the peptide active agent, aiding its passage into and through the intestinal mucus, (3) enhancing the ability of the normally polar peptide active agent to cross the epithelial barrier of the brush border membrane; and (4) increasing transcellular or paracellular transport as described above.
  • surface active agents When surface active agents are used as the absorption enhancers, it is preferred that they be free flowing powders for facilitating the mixing and loading of capsules during the manufacturing process. Because of inherent characteristics of salmon calcitonin and other peptides (e.g., their isoelectric point, molecular weight, amino acid composition, etc.) certain surface active agents interact best with certain peptides. Indeed, some can undesirably interact with the charged portions of salmon calcitonin and prevent its absorption, thus undesirably resulting in decreased bioavailability.
  • any surface active agent used as an absorption enhancer be selected from the group consisting of (i) anionic surface active agents that are cholesterol derivatives (e.g., bile acids), (ii) cationic surface agents (e.g., acyl carnitines, phospholipids and the like) , (iii) non-ionic surface active agents, and (iv) mixtures of anionic surface active agents (especially those having linear hydrocarbon regions) together with negative charge neutralizers .
  • Negative charge neutralizers include but are not limited to acyl carnitines, cetyl pyridinium chloride, and the like. It is also preferred that the absorption enhancer be soluble at acid pH, particularly in the 3.0 to 5.0 range.
  • One especially preferred combination that has worked well with salmon calcitonin mixes cationic surface active agents with anionic surface active agents that are cholesterol derivatives, both of which are soluble at acid pH.
  • a particularly preferred combination is an acid soluble bile acid together with a cationic surface active agent.
  • An acyl carnitine and sucrose ester is a good combination. When a particular absorption enhancer is used alone, it is preferred that it be a cationic surface active agent.
  • Acyl carnitines e.g. , lauroyl carnitine
  • phospholipids and bile acids are particularly good absorption enhancers, especially acyl carnitine.
  • Anionic surfactants that are cholesterol derivatives are also used in some embodiments. It is the intent of these preferences to avoid interactions with the peptide agent that interfere with absorption of peptide agent into the blood.
  • preferred detergents when used as the absorption enhancers of the invention, are either biodegradable or reabsorbable (e.g. biologically recyclable compounds such as bile acids, phospholipids, and/or acyl carnitines) , preferably biodegradable.
  • biodegradable or reabsorbable e.g. biologically recyclable compounds such as bile acids, phospholipids, and/or acyl carnitines
  • Acylcarnitines are believed particularly useful in enhancing paracellular transport .
  • a bile acid or another anionic detergent lacking linear hydrocarbons
  • a cationic detergent is used in combination with a cationic detergent, salmon calcitonin is better transported both to and through the intestinal wall .
  • Preferred absorption enhancers include: (a) salicylates such as sodium salicylate, 3- methoxysalicylate, 5-methoxysalicylate and homovanilate; (b) bile acids such as taurocholic, tauorodeoxycholic, deoxycholic, cholic, glycholic, lithocholate, chenodeoxycholic , ursodeoxycholic , ursocholic, dehydrocholic, fusidic, etc.; (c) non-ionic surfactants such as polyoxyethylene ethers (e.g. Brij 36T, Brij 52, Brij 56, Brij 76, Brij 96 , Texaphor A6, Texaphor A14,
  • salicylates such as sodium salicylate, 3- methoxysalicylate, 5-methoxysalicylate and homovanilate
  • bile acids such as taurocholic, tauorodeoxycholic, deoxycholic, cholic, glycholic,
  • Texaphor A60 etc. p-t-octyl phenol polyoxyethylenes (Triton X-45, Triton X-100, Triton X-114, Triton X-305 etc.) nonylphenoxypoloxyethylenes (e.g. Igepal CO series), polyoxyethylene sorbitan esters (e.g.
  • Tween-20, Tween-80 etc. anionic surfactants such as dioctyl sodium sulfosuccinate;
  • anionic surfactants such as dioctyl sodium sulfosuccinate;
  • lyso-phospholipids such as lysolecithin and lysophosphatidylethanolamine;
  • acylcarnitines, acylcholines and acyl amino acids such as lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, lauroylcholine, myristoylcholine, palmitoylcholine, hexadecyllysine, N-acylphenylalanine, N-acylglycine etc .
  • medium-chain glycerides which are mixtures of mono-, di- and triglycerides containing medium-chain-length fatty acids
  • caprylic, capric and lauric acids (caprylic, capric and lauric acids); (i) ethylene- diaminetetraacetic acid; (j) cationic surfactants such as cetylpyridinium chloride; (k) fatty acid derivatives of polyethylene glycol such as Labrasol, Labrafac, etc.; and
  • alkylsaccharides such as lauryl maltoside, lauroyl sucrose, myristoyl sucrose, palmitoyl sucrose, etc.
  • cationic ion exchange agents e.g. detergents
  • they may prevent the binding of salmon calcitonin or other peptide active agents to mucus.
  • Preferred cationic ion exchange agents include protamine chloride or any other polycation.
  • a water-soluble barrier separate the protease inhibitors and/or the pH-lowering agent from the acid resistant protective vehicle.
  • a conventional pharmaceutical capsule can be used for the purpose of providing this barrier.
  • Many water soluble barriers are known in the art and include, but are not limited to, hydroxypropyl methylcellulose and conventional pharmaceutical gelatins.
  • another peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the peptide is preferably from 1.0 to 10.0 percent by weight relative to the weight of the overall pharmaceutical composition (excluding protective vehicle) .
  • this second peptide is not physiologically active and is most preferably a food peptide such as soy bean peptide or the like.
  • this second peptide may also increase bioavailability by acting as a protease scavenger that desirably competes with the peptide active agent for protease interaction.
  • the second peptide may also aid the active compound's passage through the liver.
  • compositions of the invention may optionally also include common pharmaceutical diluents, glidents, lubricants, gelatin capsules, preservatives, colorants and the like in their usual known sizes and amounts .
  • Any carrier or vehicle that protects the salmon calcitonin from stomach proteases and then dissolves so that the other ingredients of the invention may be released in the intestine is suitable.
  • Many such enteric coatings are known in the art, and are useful in accordance with the invention. Examples include cellulose acetate phthalate, hydroxypropyl methylethylcellulose succinate, hydroxypropyl methylcellulose phthalate, carboxyl methylethylcellulose and methacrylic acid-methyl methacrylate copolymer.
  • the active peptide, absorption enhancers such as solubility and/or uptake enhancer (s), and pH-lowering compound (s), are included in a sufficiently viscous protective syrup to permit protected passage of the components of the invention through the stomach.
  • enteric coatings for protecting the peptide agent from stomach proteases may be applied, for example, to capsules after the remaining components of the invention have been loaded within the capsule.
  • enteric coating is coated on the outside of a tablet or coated on the outer surface of particles of active components which are then pressed into tablet form, or loaded into a capsule, which is itself preferably coated with an enteric coating.
  • the carrier or vehicle it is very desirable that all components of the invention be released from the carrier or vehicle, and solubilized in the intestinal environment as simultaneously as possible. It is preferred that the vehicle or carrier release the active components in the small intestine where uptake enhancers that increase transcellular or paracellular transport are less likely to cause undesirable side effects than if the same uptake enhancers were later released in the colon. It is emphasized, however, that the present invention is believed effective in the colon as well as in the small intestine. Numerous vehicles or carriers, in addition to the ones discussed above, are known in the art. It is desirable (especially in optimizing how simultaneously the components of the invention are released) to keep the amount of enteric coating low.
  • the enteric coating adds no more than 30% to the weight of the remainder of pharmaceutical composition (the "remainder” being the pharmaceutical composition exclusive of enteric coating itself) . More preferably, it adds less than 20%, especially from 12% to 20% to the weight of the uncoated composition.
  • the enteric coating preferably should be sufficient to prevent breakdown of the pharmaceutical composition of the invention in 0. IN HCl for at least two hours, then capable of permitting complete release of all contents of the pharmaceutical composition within thirty minutes after pH is increased to 6.3 in a dissolution bath in which said composition is rotating at 100 revolutions per minute.
  • the total weight of all pH-lowering agents and/or protease inhibitors and the total weight of all absorption enhancers in a given pharmaceutical composition is included in the foregoing preferred ratios. For example, if a pharmaceutical composition includes two pH-lowering agents and three absorption enhancers, the foregoing ratios will be computed on the total combined weight of both pH-lowering agents and the total combined weight of all three absorption enhancers.
  • the pharmaceutical composition comprises granules that include a pharmaceutical binder having the peptide active agent, the pH-lowering agent and the absorption enhancer uniformly dispersed within said binder.
  • Preferred granules may also consist of an acid core, surrounded by a uniform layer of organic acid, a layer of enhancer and a layer of peptide that is surrounded by an outer layer of organic acid.
  • Granules may be prepared from an aqueous mixture consisting of pharmaceutical binders such as polyvinyl pyrrolidone or hydroxypropyl methylcellulose, together with the pH- lowering agents, absorption enhancers and peptide active agents of the invention.
  • pharmaceutical binders such as polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • a preferred pharmaceutical composition of the invention includes a size 00 gelatin capsule filled with 0.25 mg. of salmon calcitonin linked to an MT, 400 mg. of granular citric acid (available for example from Archer Daniels Midland Corp.), 50 mg. of taurodeoxycholic acid (available for example from SIGMA) , 50 mg. lauroyl carnitine (SIGMA) .
  • All of the ingredients are preferably for eventual insertion into the gelatin capsule, and are preferably powders which may be added to a blender in any order. Thereafter, the blender is run for about three minutes until the powders are thoroughly intermixed. Then the mixed powders are loaded into the large end of the gelatine capsules. The other end of the capsule is then added, and the capsule snapped shut. 500 or more such capsules may be added to a coating device (e.g., Vector LDCS 20/30 Laboratory Development Coating System (available from Vector Corp., Marion, Iowa)) .
  • a coating device e.g., Vector LDCS 20/30 Laboratory Development Coating System (available from Vector Corp., Marion, Iowa)
  • An enteric coating solution is made as follows.
  • EUDRAGIT L30 D-55 a methacrylic acid copolymer with methacylic acid methyl ester, an enteric coating available from ROHM Tech Inc., Maidan, Mass.
  • The-' capsules are weighed and placed into the drum of the coating machine.
  • the machine is turned on to rotate the drum (now containing capsules) at 24-28 rpm.
  • the temperature of inlet sprayer is preferably about 45°C. Exhaust temperatures are preferably about 30°C.
  • Uncoated capsule temperature is preferably about 25 °C. Air flow is about 38 cubic feet per minute.
  • a tube from the machine is then inserted into the coating solution prepared " as discussed above.
  • the pump is then turned on for feeding solution into the coating device. Coating then proceeds automatically.
  • the machine can be stopped at any time to weigh capsules to determine if the coating amount is sufficient. Usually coating is allowed to proceed for 60 minutes.
  • the pump is then turned off for about five minutes while the machine is still running to help dry the coated capsules .
  • the machine can then be turned off.
  • the capsule coating is then complete, although it is recommended that the capsules be air dried for about two days.
  • the concentration of expensive salmon calcitonin in the pharmaceutical preparation of the invention may be kept relatively low. Specific formulation examples are set forth in examples infra .
  • Nasally administered salmon calcitonin (200 International units) has proven effective against osteoporosis at peak levels as low as 10 picograms per milliliter. Some patients report some gastrointestinal distress at high peak levels (e.g. at or above 200 picograms per milliliter) . Accordingly, it is preferred that serum salmon calcitonin peak between 10 and 150 picograms per milliliter, more preferably between 10 and 50 picograms per milliliter. The serum levels may be measured by radioimmunoassay techniques known in the art . The attending physician may monitor patient response, salmon calcitonin blood levels, or surrogate markers of bone disease (such as urinary pyridinoline or deoxypyridinoline) , especially during the initial phase of treatment (1-6 months) .
  • bone disease such as urinary pyridinoline or deoxypyridinoline
  • the bioavailability achievable in accordance with the present invention permits oral delivery of salmon calcitonin into the blood at the above-identified preferred concentration levels while using only 10-1000 micrograms of salmon calcitonin per capsule, preferably 10-400 micrograms, especially between 10 and 200 micrograms .
  • a single capsule be used at each administration because a single capsule best provides simultaneous release of the polypeptide, pH- lowering agent and absorption enhancers. This is highly desirable because the acid is best able to reduce undesirable proteolytic attack on the polypeptide when the acid is released in close time proximity to release of the polypeptide. Near simultaneous release is best achieved by administering all components of the invention as a single pill or capsule. However, the invention also includes, for example, dividing the required amount of acid and enhancers, when used, among two or more capsules which may be administered together such that they together provide the necessary amount of all ingredients. "Pharmaceutical composition, " as used herein includes a complete dosage appropriate to a particular administration to a human patient regardless of how it is subdivided so long as it is for substantially simultaneous administration.
  • Example 1 Preparation of A Purified Fusion Peptide of Salmon Calcitonin (sCT) and Enzvme-Cleavable Membrane Translocator (MT) 1. Construction of Fusion Peptide of Salmon Calcitonin
  • a membrane translocator sequence corresponding to the Protein Transduction Domain of the HIV TAT protein was used, The amino acid sequence of this MT is as follows :
  • the TRP-VAL-ALA sequence is enzyme-cleavable first by endothelin converting enzyme between TRP and VAL and the resulting VAL-ALA-sCT-GLY sequence is cleavable by dipeptidyl-peptidase IV to liberate authentic sCT-Gly.
  • Oligos 1 and 2 encode the MT sequence plus an amino acid at the 5' end for ligation into the appropriate vector, the enzyme cleavable sequence, and the first 4 amino acids of the sCT-Gly sequence (CYS-SER-ASN-LEU)
  • the gene for sCT-Gly was obtained by PCR amplification of the appropriate region from plasmid, psCT025, which is a previously constructed expression plasmid containing the sCT gene (see Figure 3) , or from any other suitable source of the sCT gene.
  • the primers used for the PCR amplification generated a 125 bp amplified fragment that contains the entire sCT gene.
  • PCR amplification was done using a commercially available kit from Boehringer Mannheim. The PCR cycling was done as follows:
  • the 5' -PCR primer contains an Ace I site, and the 3' primer contains a Nco I site.
  • the 125 bp PCR fragment was digested with Ace I and the resulting 100 bp fragment was isolated from an agarose gel and quantified.
  • the double stranded DNA fragment formed by ligation of oligos 1 and 2 was now ligated to the 100 bp PCR fragment at the common Ace I site.
  • the product of this ligation was digested with Nco I.
  • the resulting 150 bp DNA sequence had a blunt 5' end and a Nco I compatible 3' end. This DNA fragment, designated MT3sCT01, was gel purified and quantified.
  • the universal cloning vector pUSEC-05 ( Figure 1) , contains dual Tac and Lac promoters, followed by a Shine- Delgarno sequence and the sequence for the omp A signal peptide. Downstream of the omp A signal are cloning sites for Stu I, Nco I, and Sty I.
  • pUSEC-05 was linearized by digestion with Stu I and Nco I and MT3sCT01 sequence was ligated into the vector at blunt ends on the 5' end and the Nco I site on the 3' end to create the plasmid pMT3sCT01 ( Figure 2) .
  • the MT3sCT01 plasmid was characterized by restriction digest mapping and sequencing to confirm the presence of all relevant genes and the sequence of the cloned DNA fragment .
  • This plasmid was used to transform the host strain E. coli BLR to create the recombinant expression strain UGL286.
  • the universal cloning vector pUSEC-06 ( Figure 4) , contains dual Tac and Lac promoters, followed by a Shine- Delgarno sequence and the sequence for the ompA signal peptide.
  • the Multiple Cloning Site [MCS] of the pUSECO ⁇ plasmid vector contains the restriction enzyme sites for BspEl and Asc 1
  • the pUSEC-06 was linearized by digestion with these enzymes, creating sticky ends for directional ligation.
  • the plasmid, pMT3sCT01 was cut with BspEl and Asc 1 as well, and the 713 bp DNA fragment containing the MT3sCT01 DNA sequence was isolated, gel purified, and quantified.
  • the MT3sCT01 sequence was directionally ligated into the vector with the BspEl at the 5' end and the Ascl site on the 3 ' end to create the plasmid, pMT3- sCT-02 ( Figure 5) .
  • the pMT-sCT-02 plasmid was linearized with 2 other restriction enzymes, Aflll and Mfe I, with sites within the MCS immediately adjacent to each other.
  • the pMTsCTOl plasmid was also cut with the same two enzymes to release the DNA fragment MT3sCT01.
  • the isolated, gel purified DNA fragment was directionally cloned into the vector at the Afl Il/Mfe compatible ends to create the plasmid pMT3-sCT-03 ( Figure 6) , which encodes tandem MT3sCT01 sequences, as well as the kanamycin resistance gene, the lacl Q gene, and the secretion factor genes, secE and prlA4.
  • the pMT3-sCT-03 plasmid was used to transform competent E. coli BLR cells to create the recombinant expression strain, UGL 716.
  • the expression strain, UGL 716 was grown in bench scale fermentation according to Series 700 Fermentation Protocol, ref. CPM:022:035, under standard conditions of pH, d ⁇ 2 , temperature and feeding/induction regime. The fermentation was harvested by centrifugation at 26 hours and the cells were stored at -20C until lysis. The fermentation samples were evaluated by DNA analysis and protein expression analysis .
  • DNA analysis Restriction enzyme mapping analysis of the plasmid prepared from the fermentation samples verified the presence of pMT3sCT03 plasmid and all relevant genes.
  • E. coli inclusion bodies were partially solublized with 800 L 0.1 M HCl, 5 M guanidine HCl. The suspension was centrifuged at 20,000 rpm (50,000 x g) for 60 minutes. The resultant supernatant was collected and loaded directly onto a Vydac C18 (22 x 250 mm) , 10 m, 300 column equilibrated with 0.1% trifluoroacetic acid. The column was subjected to a linear gradient from 100% A (0.1% TFA) to 100 % B (0.1% trifluoroacetic, 80% acetonitrile) over 90 minutes. The column was operated at 25 mL/min.
  • the putative MTS3sCTgly recombinant protein was in the insoluble fraction.
  • the migration was not consistent with a predicted size of ⁇ 5kDa for the MTS3sCTgly peptide, instead, the r-protein on the gel was migrating nearly coincident with the ⁇ 15kDa marker protein.
  • the fractionation and the gel analysis data also suggested that the signal peptide might be unprocessed in this construct, and this is corroborated by the data from amino acid analysis and mass spectroscopy.
  • the total size of a protein composed of unprocessed OmpA/MTS3/sCTgly would be ⁇ 7KDa.
  • Example 2 Effect of OmpA-MT3 on the Absorption of Salmon Calcitonin from Rat Duodenum
  • the cannula was fitted to a three way valve through which blood was sampled and replaced with physiological saline containing heparin.
  • Example 3 Effect of the HIV TAT Protein Transduction Domain as an MT on the Absorption of Salmon Calcitonin from Doq Duodenum
  • Two formulations are used to test the efficacy of MT3-SCT-01 fusion peptide.
  • the first formulation (FI) is prepared by blending 13 g citric acid, 1.3 g lauroylcarnitine, 0.65 g talc and
  • the other formulation (F2) is prepared by blending the same mixture except that sCT is replaced with an equivalent amount of
  • MT3sCT01 Both blends are used to fill size 00 gelatin capsules, and the capsules are coated with Eudragit L30D- 55.
  • the resulting enteric-coated capsules contain approximately 1 to 2 mg of either sCT (FI) or MT3sCT01
  • the amount of sCT in plasma samples of dogs given either of the two formulations is measured by radioimmunoassay (RIA) using a commercially available kit .
  • RIA radioimmunoassay
  • the bioavailability of sCT in dogs given FI is expected to be approximately 1%, whereas the bioavailability of sCT in dogs given F2 is expected to be at least 1.2%.
  • the in vivo cleavage of MT from sCT in dogs given F2 is proven by applying samples of plasma from dogs given FI and F2 to an HPLC column and collecting the effluent in plastic tubes. The solvent in the tubes is removed under vacuum and analyzed for the presence of sCT by RIA.
  • the in vivo cleavage of MT3sCT01 is established by showing that the retention time of sCT in the plasma from dogs given F2 is the same as the retention time of sCT in the plasma of dogs given FI .

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Abstract

La biodisponibilité d'agents actifs peptidiques à administrer oralement est améliorée par une composition pharmaceutique conférant une libération ciblée du peptide dans l'intestin, ainsi que par la liaison du peptide actif à un translocateur de membrane pouvant être coupé au moins partiellement in vivo par une enzyme. La composition renferme un excipient de protection résistant aux acides qui transporte les constituants de l'invention à travers l'estomac ; et une quantité suffisante d'un agent diminuant le pH pour réduire le pH intestinal local. Tous les constituants sont libérés ensemble dans l'intestin avec le peptide.
PCT/US2001/044980 2000-11-30 2001-11-30 Apport oral ameliore de peptides au moyen de translocateurs de membrane pouvant etre coupes par une enzyme WO2002043767A1 (fr)

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JP2002545737A JP4476544B6 (ja) 2000-11-30 2001-11-30 酵素で切断可能な膜トランスロケーターを用いて改善されたペプチドの経口送達
CA2430366A CA2430366C (fr) 2000-11-30 2001-11-30 Apport oral ameliore de peptides au moyen de translocateurs de membrane pouvant etre coupes par une enzyme
ES01996021.0T ES2642628T3 (es) 2000-11-30 2001-11-30 Suministro oral mejorado de péptidos, mediante la utilización de translocadores de membrana, segmentables mediante una enzima
DK01996021.0T DK1339431T3 (en) 2000-11-30 2001-11-30 IMPROVED ORAL ADMINISTRATION OF PEPTIDES USING ENZYM SPLIT MEMBRANE TRANSLATORS
AU2706002A AU2706002A (en) 2000-11-30 2001-11-30 Improved oral delivery of peptides using enzyme-cleavable membrane translocators
EP01996021.0A EP1339431B1 (fr) 2000-11-30 2001-11-30 Apport oral amelioré de peptides au moyen de translocateurs de membrane pouvant être coupés par une enzyme

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US09/997,465 US6673574B2 (en) 2000-11-30 2001-11-29 Oral delivery of peptides using enzyme-cleavable membrane translocators

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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004064758A2 (fr) 2003-01-21 2004-08-05 Unigene Laboratories Inc. Administration amelioree de peptides par voie orale
WO2007070450A3 (fr) * 2005-12-09 2008-01-31 Unigene Lab Inc Produits pharmaceutiques peptidiques oraux a action rapide
WO2011069038A2 (fr) 2009-12-03 2011-06-09 Synergy Pharmaceuticals, Inc. Agonistes de la guanylate cyclase utiles dans le traitement de l'hypercholestérolémie, de l'athérosclérose, d'une coronaropathie, des calculs biliaires, de l'obésité et d'autres maladies cardiovasculaires
CN102740889A (zh) * 2009-12-04 2012-10-17 卫生防护机构 用于预防或抑制艰难梭菌感染的疗法
WO2013138352A1 (fr) 2012-03-15 2013-09-19 Synergy Pharmaceuticals Inc. Formulations d'agonistes de la guanylate cyclase c et procédés d'utilisation
US8709428B2 (en) 2009-02-20 2014-04-29 Health Protection Agency Antibodies to Clostridium difficile toxins
US8734849B2 (en) 2003-07-15 2014-05-27 Evonik Roehm Gmbh Multiparticle pharmaceutical dosage form containing a mucoadhesively formulated peptide or protein active substances method for producing said pharmaceutical dosage form
WO2014131024A2 (fr) 2013-02-25 2014-08-28 Synergy Pharmaceuticals Inc. Agonistes de la guanylate cyclase et applications associées
WO2014151200A2 (fr) 2013-03-15 2014-09-25 Synergy Pharmaceuticals Inc. Compositions utiles pour le traitement de troubles gastro-intestinaux
WO2014151206A1 (fr) 2013-03-15 2014-09-25 Synergy Pharmaceuticals Inc. Agonistes de la guanylate cyclase et leurs utilisations
EP2810951A2 (fr) 2008-06-04 2014-12-10 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utile dans le traitement de troubles gastro-intestinaux, d'une inflammation, d'un cancer et d'autres troubles
WO2015054649A2 (fr) 2013-10-10 2015-04-16 Synergy Pharmaceuticals, Inc. Agonistes de guanylate cyclase utiles pour le traitement de troubles induits par les opioïdes
US9040032B2 (en) 2010-04-21 2015-05-26 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable opioid prodrugs and inhibitors thereof
US9095627B2 (en) 2011-03-09 2015-08-04 Signature Therapeutics, Inc. Opioid prodrugs with heterocyclic linkers
US9139612B2 (en) 2011-03-09 2015-09-22 Signature Therapeutics, Inc. Active agent prodrugs with heterocyclic linkers
EP2998314A1 (fr) 2007-06-04 2016-03-23 Synergy Pharmaceuticals Inc. Agonistes de guanylase cyclase utiles pour le traitement de troubles gastro-intestinaux, d'inflammation, de cancer et d'autres troubles
US9399017B2 (en) 2007-05-29 2016-07-26 Enteris Biopharma, Inc. Peptide pharmaceutical for oral delivery
US9457086B2 (en) 2013-03-05 2016-10-04 Enteris Biopharma, Inc. Pharmaceuticals for oral delivery
US9493477B2 (en) 2009-09-08 2016-11-15 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable ketone-modified opioid prodrugs and optional inhibitors thereof
US9499581B2 (en) 2011-01-11 2016-11-22 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable oxycodone prodrug
WO2017123634A1 (fr) 2016-01-11 2017-07-20 Synergy Pharmaceuticals, Inc. Formulations et méthodes pour traiter la rectocolite hémorragique
EP3241839A1 (fr) 2008-07-16 2017-11-08 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utiles pour le traitement de troubles gastro-intestinaux, inflammatoires, cancéreux et autres
US9833411B2 (en) 2015-01-12 2017-12-05 Enteris Biopharma, Inc. Solid oral dosage forms
US11179355B2 (en) 2010-04-21 2021-11-23 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable amphetamine prodrugs and inhibitors thereof

Families Citing this family (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7030082B2 (en) * 2001-09-07 2006-04-18 Nobex Corporation Pharmaceutical compositions of drug-oligomer conjugates and methods of treating disease therewith
CN1838968A (zh) 2003-08-08 2006-09-27 艾伯吉尼斯公司 针对甲状旁腺激素(pth)之抗体和其用途
US7318925B2 (en) * 2003-08-08 2008-01-15 Amgen Fremont, Inc. Methods of use for antibodies against parathyroid hormone
US20050215476A1 (en) * 2004-01-21 2005-09-29 Unigene Laboratories Inc. Amidated parathyroid hormone fragments and uses thereof
CN1997660A (zh) 2004-04-21 2007-07-11 芝加哥大学 肌球蛋白轻链激酶抑制剂及其使用
US7713737B2 (en) * 2004-10-04 2010-05-11 Trinity Biosystems, Inc. Methods and compositions for needleless delivery of macromolecules
EP1863850B1 (fr) * 2005-03-30 2009-09-23 Jung Moon Kim Polypeptides non actives possedant une fonction de regeneration tissulaire et methode de preparation
KR100775958B1 (ko) 2005-03-30 2007-11-13 김정문 조직재생 기능을 가지는 비활성 폴리펩티드 및 그 제조방법
AU2006288703B2 (en) * 2005-09-06 2011-09-22 Oramed Pharmaceuticals, Inc. Methods and compositions for oral administration of proteins
US7666991B2 (en) * 2005-12-05 2010-02-23 Trinity Biosystems, Inc. Compositions for needleless delivery of antibodies
EP1971367A4 (fr) * 2005-12-05 2010-04-07 Trinity Biosystems Inc Procedes et compositions d'apport sans aiguille de partenaires de liaison
WO2007109110A2 (fr) 2006-03-16 2007-09-27 Trinity Biosystems, Inc. Procédés permettant d'augmenter la taille d'animaux au moyen de constructions d'apport sans aiguille
US20090092660A1 (en) * 2006-08-09 2009-04-09 Trinity Biosystems, Inc. Methods and compositions for needleless delivery of particles
WO2008084899A1 (fr) * 2007-01-10 2008-07-17 Insilicotech Co., Ltd. Peptide d'entérocyte transcytotique de ciblage de tissu et de cellule m identifié au moyen d'un procédé d'affichage de phages et système de distribution de médicaments utilisant ledit peptide
US20100119528A1 (en) * 2008-01-17 2010-05-13 Gobinda Sarkar Transport of Biologically Active Molecules into a Cell, Mitochondrion, or Nucleus
BRPI0907077A2 (pt) * 2008-03-26 2016-07-26 Oramed Ltd métodos e composições para administração oral de proteínas
CA2723434C (fr) 2008-05-05 2018-01-23 Oramed Ltd. Procedes et compositions pour l'administration d'exenatide par voie orale
EP2762150A1 (fr) 2009-03-12 2014-08-06 Nordic Bioscience A/S Traitement des diabètes et du syndrome métabolique
BR112012000908A2 (pt) 2009-07-14 2019-09-24 Mayo Found Medical Education & Res liberação de agentes ativos através da barreira hematoencefálica mediada por peptídeo em associação não covalente
WO2011041494A1 (fr) * 2009-09-30 2011-04-07 Baylor College Of Medicine Peptides tex14 comme nouveaux agents anti-tumoraux
RU2593715C2 (ru) 2010-09-15 2016-08-10 Рэндэлл Дж. МРСНИ Системы и способы доставки биоактивных средств с применением транспортных последовательностей, происходящих от бактериального токсина
US11246915B2 (en) 2010-09-15 2022-02-15 Applied Molecular Transport Inc. Cholix toxin-derived fusion molecules for oral delivery of biologically active cargo
EP3095484B1 (fr) 2011-11-02 2018-05-02 KeyBioscience AG Mimétiques de calcitonine pour le traitement de maladies et de troubles
KR102019911B1 (ko) 2011-11-02 2019-09-09 키바이오사이언스 아게 질병 및 장애 치료용 펩타이드 유사체
JP6174601B2 (ja) 2012-01-03 2017-08-02 オラムド エルティーディー． 糖尿病を処置するための方法及び組成物
JP6567827B2 (ja) 2012-02-01 2019-08-28 オラムド エルティーディー． プロテアーゼ阻害剤を含有する組成物、該組成物を含む組成物、および、該組成物を製造および使用する方法
US20130338080A1 (en) * 2012-05-24 2013-12-19 The Trustees Of The University Of Pennsylvania Compositions and methods for treating an activated b-cell diffuse large b-cell lymphoma
BR112015006176B1 (pt) 2012-09-21 2023-04-18 Intensity Therapeutics, Inc Uso de um agente terapêutico e de um agente de intensificação de permeação intracelular
CN110151979A (zh) 2013-01-03 2019-08-23 奥拉姆德有限公司 用于治疗nafld、肝性脂肪变性及其后遗症的方法和组合物
US9840533B2 (en) 2013-04-29 2017-12-12 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
RS57152B1 (sr) 2013-11-14 2018-07-31 Keybioscience Ag Mimetici kalcitonina za lečenje bolesti i poremećaja
EP3119415A4 (fr) 2014-03-07 2017-11-29 The Arizona Board of Regents on behalf of the University of Arizona Peptides crmp2 non narcotiques ciblant des canaux sodiques pour la douleur chronique
ES2900328T3 (es) 2014-05-07 2022-03-16 Applied Molecular Transport Inc Moléculas de fusión derivadas de toxina Cholix para la administración oral de carga biológicamente activa
GB201500263D0 (en) 2015-01-08 2015-02-25 Keybioscience Ag Calcitonin analogues for treating diseases and disorders
US10653749B2 (en) 2015-04-16 2020-05-19 The Regents Of The University Of California Targeting GIV-GEF-GI signaling for treating diverse diseases
US10072065B2 (en) 2015-08-24 2018-09-11 Mayo Foundation For Medical Education And Research Peptide-mediated delivery of immunoglobulins across the blood-brain barrier
US12186365B2 (en) * 2015-11-17 2025-01-07 Seoul National University R&Db Foundation Composition for regulating cell division comprising FCHO1 modulator, and method for regulating cell division using same
GB201704429D0 (en) 2017-03-21 2017-05-03 Keybioscience Ag Calcitonin mimetics for treating diseases and disorders
GB201707955D0 (en) 2017-05-18 2017-07-05 Keybioscience Ag Dual amylin and calcitonin receptor agonists for treating diseases and disorders
DK3762009T3 (da) 2018-03-08 2022-06-20 Applied Molecular Transport Inc Toxin-afledte indgivelseskonstrukter til oral indgivelse
WO2019173787A1 (fr) 2018-03-08 2019-09-12 Applied Molecular Transport Inc. Constructions d'administration dérivées de toxines pour administration orale
GB201813677D0 (en) 2018-08-22 2018-10-03 Keybioscience Ag Calcitonin mimetics for treating diseases and disorders
GB201813678D0 (en) 2018-08-22 2018-10-03 Keybioscience Ag Acylated calcitonin mimetics
BR112021009003A2 (pt) 2018-11-07 2021-08-17 Applied Molecular Transport Inc. construtos de liberação para transcitose e métodos relacionados.
EP3826682A4 (fr) 2018-11-07 2021-11-17 Applied Molecular Transport Inc. Supports dérivés de cholix pour administration orale de chargement hétérologue
EP3844169A4 (fr) 2019-08-16 2021-12-15 Applied Molecular Transport Inc. Compositions, formulations et production et purification d'interleukines

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034295A1 (fr) 1994-06-13 1995-12-21 Vanderbilt University Nouveau procede destine a importer des molecules biologiquement actives dans des cellules
WO1997033531A1 (fr) 1996-03-15 1997-09-18 Unigene Laboratories, Inc. Produits pharmaceutiques peptidiques oraux
US5766620A (en) * 1995-10-23 1998-06-16 Theratech, Inc. Buccal delivery of glucagon-like insulinotropic peptides
US5968895A (en) * 1996-12-11 1999-10-19 Praecis Pharmaceuticals, Inc. Pharmaceutical formulations for sustained drug delivery
US6248558B1 (en) * 1998-03-31 2001-06-19 Vanderbilt University Sequence and method for genetic engineering of proteins with cell membrane translocating activity
WO2001044199A1 (fr) * 1999-12-16 2001-06-21 Emisphere Technologies, Inc. Composes et compositions facilitant l'administration d'agents actifs

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674980A (en) * 1989-12-21 1997-10-07 Biogen Inc Fusion protein comprising tat-derived transport moiety
US6030613A (en) * 1995-01-17 2000-02-29 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
EP1086126A1 (fr) * 1998-06-10 2001-03-28 The Queen's University of Belfast Peptide permeable aux cellules
CA2386231C (fr) * 1999-09-27 2010-09-14 Daniel J. O'mahony Systeme de relargage de medicaments a base d'un peptide de translocation membranaire

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034295A1 (fr) 1994-06-13 1995-12-21 Vanderbilt University Nouveau procede destine a importer des molecules biologiquement actives dans des cellules
US5766620A (en) * 1995-10-23 1998-06-16 Theratech, Inc. Buccal delivery of glucagon-like insulinotropic peptides
WO1997033531A1 (fr) 1996-03-15 1997-09-18 Unigene Laboratories, Inc. Produits pharmaceutiques peptidiques oraux
US5968895A (en) * 1996-12-11 1999-10-19 Praecis Pharmaceuticals, Inc. Pharmaceutical formulations for sustained drug delivery
US6248558B1 (en) * 1998-03-31 2001-06-19 Vanderbilt University Sequence and method for genetic engineering of proteins with cell membrane translocating activity
WO2001044199A1 (fr) * 1999-12-16 2001-06-21 Emisphere Technologies, Inc. Composes et compositions facilitant l'administration d'agents actifs

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BERNKOP-SCHNURCH, A.: "The use of inhibitory agent to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins", J. CONTROLLED RELEASE, vol. 52, March 1998 (1998-03-01), pages 1 - 16, XP004113649 *
PATEL, D. ET AL.: "Peptide targeting and delivery across the blood-brain barrier utilizing synthetic triglyceride esters: design, synthesis and bioactivity", BIOCONJUGATE CHEM., vol. 8, 1997, pages 434 - 441, XP002908641 *
See also references of EP1339431A4

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120183582A1 (en) * 2003-01-21 2012-07-19 Mehta Nozer M Oral Delivery of Peptides
WO2004064758A3 (fr) * 2003-01-21 2005-01-20 Unigene Lab Inc Administration amelioree de peptides par voie orale
JP2006517542A (ja) * 2003-01-21 2006-07-27 ユニジーン・ラボラトリーズ・インコーポレーテッド 改善された経口ペプチド送達
AU2004206949B2 (en) * 2003-01-21 2009-07-30 Enteris Biopharma, Inc. Improved oral delivery of peptides
WO2004064758A2 (fr) 2003-01-21 2004-08-05 Unigene Laboratories Inc. Administration amelioree de peptides par voie orale
US8088734B2 (en) 2003-01-21 2012-01-03 Unigene Laboratories Inc. Oral delivery of peptides
US8734849B2 (en) 2003-07-15 2014-05-27 Evonik Roehm Gmbh Multiparticle pharmaceutical dosage form containing a mucoadhesively formulated peptide or protein active substances method for producing said pharmaceutical dosage form
CN101394841B (zh) * 2005-12-09 2012-04-18 尤尼金实验室公司 快速起作用的口服肽药品
US8093207B2 (en) 2005-12-09 2012-01-10 Unigene Laboratories, Inc. Fast-acting oral peptide pharmaceutical products
WO2007070450A3 (fr) * 2005-12-09 2008-01-31 Unigene Lab Inc Produits pharmaceutiques peptidiques oraux a action rapide
US9399017B2 (en) 2007-05-29 2016-07-26 Enteris Biopharma, Inc. Peptide pharmaceutical for oral delivery
EP2998314A1 (fr) 2007-06-04 2016-03-23 Synergy Pharmaceuticals Inc. Agonistes de guanylase cyclase utiles pour le traitement de troubles gastro-intestinaux, d'inflammation, de cancer et d'autres troubles
EP2810951A2 (fr) 2008-06-04 2014-12-10 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utile dans le traitement de troubles gastro-intestinaux, d'une inflammation, d'un cancer et d'autres troubles
EP3241839A1 (fr) 2008-07-16 2017-11-08 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utiles pour le traitement de troubles gastro-intestinaux, inflammatoires, cancéreux et autres
US8709428B2 (en) 2009-02-20 2014-04-29 Health Protection Agency Antibodies to Clostridium difficile toxins
US10028945B2 (en) 2009-09-08 2018-07-24 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable ketone-modified opioid prodrugs and optional inhibitors thereof
US9493477B2 (en) 2009-09-08 2016-11-15 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable ketone-modified opioid prodrugs and optional inhibitors thereof
WO2011069038A2 (fr) 2009-12-03 2011-06-09 Synergy Pharmaceuticals, Inc. Agonistes de la guanylate cyclase utiles dans le traitement de l'hypercholestérolémie, de l'athérosclérose, d'une coronaropathie, des calculs biliaires, de l'obésité et d'autres maladies cardiovasculaires
EP2923706A1 (fr) 2009-12-03 2015-09-30 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utiles pour le traitement de l'hypercholestérolémie
US8921529B2 (en) 2009-12-04 2014-12-30 Health Protection Agency Therapies for preventing or suppressing Clostridium difficile infection
CN102740889A (zh) * 2009-12-04 2012-10-17 卫生防护机构 用于预防或抑制艰难梭菌感染的疗法
US9040032B2 (en) 2010-04-21 2015-05-26 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable opioid prodrugs and inhibitors thereof
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US9585963B2 (en) 2010-04-21 2017-03-07 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable opioid prodrugs and inhibitors thereof
US9499581B2 (en) 2011-01-11 2016-11-22 Signature Therapeutics, Inc. Compositions comprising enzyme-cleavable oxycodone prodrug
US9095627B2 (en) 2011-03-09 2015-08-04 Signature Therapeutics, Inc. Opioid prodrugs with heterocyclic linkers
US9139612B2 (en) 2011-03-09 2015-09-22 Signature Therapeutics, Inc. Active agent prodrugs with heterocyclic linkers
EP4309673A2 (fr) 2012-03-15 2024-01-24 Bausch Health Ireland Limited Formulations d'agonistes de guanylate cyclase c et leurs procédés d'utilisation
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WO2013138352A1 (fr) 2012-03-15 2013-09-19 Synergy Pharmaceuticals Inc. Formulations d'agonistes de la guanylate cyclase c et procédés d'utilisation
WO2014131024A2 (fr) 2013-02-25 2014-08-28 Synergy Pharmaceuticals Inc. Agonistes de la guanylate cyclase et applications associées
EP3718557A2 (fr) 2013-02-25 2020-10-07 Bausch Health Ireland Limited Agoniste du récepteur de la guanylate cyclase sp-333 à utiliser lors du nettoyage du côlon
US9457086B2 (en) 2013-03-05 2016-10-04 Enteris Biopharma, Inc. Pharmaceuticals for oral delivery
US9744140B2 (en) 2013-03-05 2017-08-29 Enteris Biopharma, Inc. Pharmaceuticals for oral delivery
US9526785B2 (en) 2013-03-05 2016-12-27 Enteris Biopharma, Inc. Pharmaceuticals for oral delivery
WO2014151200A2 (fr) 2013-03-15 2014-09-25 Synergy Pharmaceuticals Inc. Compositions utiles pour le traitement de troubles gastro-intestinaux
WO2014151206A1 (fr) 2013-03-15 2014-09-25 Synergy Pharmaceuticals Inc. Agonistes de la guanylate cyclase et leurs utilisations
WO2015054649A2 (fr) 2013-10-10 2015-04-16 Synergy Pharmaceuticals, Inc. Agonistes de guanylate cyclase utiles pour le traitement de troubles induits par les opioïdes
US9833411B2 (en) 2015-01-12 2017-12-05 Enteris Biopharma, Inc. Solid oral dosage forms
WO2017123634A1 (fr) 2016-01-11 2017-07-20 Synergy Pharmaceuticals, Inc. Formulations et méthodes pour traiter la rectocolite hémorragique

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EP1339431A4 (fr) 2005-10-12
AU2706002A (en) 2002-06-11
EP1339431A1 (fr) 2003-09-03
US6673574B2 (en) 2004-01-06
EP1339431B1 (fr) 2017-06-28
JP4476544B2 (ja) 2010-06-09
CA2430366A1 (fr) 2002-06-06
WO2002043767A8 (fr) 2003-04-10
US20030118610A1 (en) 2003-06-26
JP2004521874A (ja) 2004-07-22
ES2642628T3 (es) 2017-11-17
CA2430366C (fr) 2010-09-28

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